Apoptosis is a morphologically distinct form of programmed cell death that plays important roles in development, tissue homeostasis and a wide variety of diseases, including cancer, AIDS, stroke, myopathies and various neurodegenerative disorders. It is now clear that apoptosis occurs by activating an intrinsic cell suicide program which is constitutively expressed in most animal cells, and that key components of this program have been conserved in evolution from worms to insects to man. A central step in the execution of apoptosis is the activation of a specific class of cysteine proteases, termed caspases, that are widely expressed as inactive zymogens. The overall objective of the proposed research is to gain insight into the molecular mechanisms that control caspase activation and cell death. Work during the previous project period has demonstrated that a set of ubiquitin pathway proteins play a complex but specific role in regulating the onset of apoptosis via selective protein degradation. In particular, Inhibitor of Apoptosis Proteins (IAPs) can ubiquitinate certain caspases in live cells, but auto-ubiquitinate and self-destruct in cells that are doomed to die. The specific goals of this proposal are to define the mechanism by which Reaper-family protein stimulate the auto-ubiquitination of IAPs in both Drosophila and mammalian cells, and the role of this pathway for the regulation of cell death in both normal and cancer cells. Furthermore, we will test the hypothesis that defects in IAP self-conjugation contribute to cell immortalization and malignancy. For this project, we will use a multidisciplinary approach that integrates Drosophila genetics, biochemistry, cell biology, and mammalian cell culture studies. As part of this study, we propose to generate small, cell permeable peptide derivatives of Reaper ("Reaper-mimetics') to inactivate cellular IAPs ("protein knock-outs") and examine their role in the regulation of mammalian cell death. This work should significantly advance our understanding of how apoptosis is regulated, and how this process can be manipulated for therapeutic purposes.